The Causal Role of Mitochondrial Dynamics in Regulating Insulin Resistance in Diabetes: Link through Mitochondrial Reactive Oxygen Species
Insulin resistance of cybrid B4 was improved by mdivi-1, which suppresses mitochondrial fission and promotes fusion. 10 μM mdivi-1 was used to inhibit mitochondrial fission. For probing cellular insulin resistance, cybrid B4 was starved of FBS for 16 h, then incubated with mdivi-1 for 24 h, and finally treated with 0, 0.1, or 1 μM insulin for 1 h. (a) The action of Drp1 inhibitor, mdivi-1, was determined using level of p-Drp1(S616) and Drp-1. β-Actin served as loading control. (b) Mitochondrial morphology was visualized by transfecting cox4-DsRed (red fluorescence). An enlarged segment of each image was shown by a lower right square. (c-d) The MicroP algorithm categorized mitochondrial morphology into six types: small globe (blue), large globe (yellow), simple tube (green), twisted tube (orange), donut (red), and branching tube (purple). N = 75–400 mitochondria from 15–30 cells and three independent experiments. (e) p-IRS1-1(Y896), IRS-1, p-AKT(S473), and AKT were determined using Western blotting. β-Actin served as loading control. (f) Abundance of GLUT1 and GLUT4 in cytoplasm and membrane subfractionation was probed using Western blotting. β-Actin and Na+-K+ATPase served as loading control of cytoplasm and membrane fraction. The quantitative result (mean ± SEM) of Western blotting was calculated from at least three independent experiments. .
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